Ca2+/calmodulin-dependent protein kinase II isoenzymes gamma and delta are both present in H+/K+-ATPase-containing rabbit gastric tubulovesicles

Abstract

Ca2+/calmodulin-dependent protein kinase II is thought to participate in M3 muscarinic receptor-mediated acid secretion in gastric parietal cells. During acid secretion tubulovesicles carrying H+/K+-ATPase fuse with the apical membrane. We localized Ca2+/calmodulin-dependent protein kinase II from highly purified rabbit gastric tubulovesicles using Ca2+/calmodulin-dependent protein kinase II isoform-specific antibodies, in vitro phosphorylation and pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II activity by the potent Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. The presence of Ca2+/calmodulin-dependent protein kinase II in tubulovesicles was shown by immunoblot detection of both Ca2+/calmodulin-dependent protein kinase II-gamma (54 kDa) and Ca2+/calmodulin-dependent protein kinase II-delta (56.5 kDa). The immunoprecipitated Ca2+/calmodulin-dependent protein kinase II from tubulovesicles showed Ca2+/calmodulin-dependent protein kinase activity by phosphorylating autocamtide-II, a specific synthetic Ca2+/calmodulin-dependent protein kinase II substrate. KN-62 inhibited the in vitro autophosphorylation of tubulovesicle-associated Ca2+/calmodulin-dependent protein kinase II (IC50 = 11 nM). During the search for potential Ca2+/calmodulin-dependent protein kinase II substrates we identified different proteins associated with tubulovesicles, such as synaptophysin and beta-tubulin immunoreactivity, which were identified using specific antibodies. These targets are known to participate in intracellular membrane traffic. Ca2+/calmodulin-dependent protein kinase II is thought to play an important role in regulating tubulovesicular motor activity and therefore in acid secretion.